JP2008136453A - Method for identifying activity-inhibitory factor for von willebrand factor-cleaving enzyme - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying the cause of the low activity level of a von Willebrand factor-cleaving enzyme, and to provide a method for determining a proper method for treating diseases stemmed from such a low activity level. <P>SOLUTION: A bilirubin level is determined for a subject where the specific activity level of a von Willebrand factor-cleaving enzyme is low, thus determining the cause of the low activity level of the enzyme, and enabling a proper method for treating diseases stemmed from such a low activity level to be determined as well. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for grasping an activity inhibitor of von Willebrand factor cleaving enzyme.

  Von Willebrand factor (vWF-cleaving protease; vWF-CP), also known as ADAMTS13 deficiency, has been identified as the etiology of familial thrombocytopenic purpura (TTP) It was revealed that ADAMTS13 activity was significantly reduced due to the mutation in this gene (Non-patent Document 1). On the other hand, more common acquired TTP has been reported to occur because ADAMTS13 activity is inhibited by autoantibodies (IgG neutralizing antibody, IgM non-neutralizing antibody) against ADAMTS13. (Non-Patent Documents 2 and 3).

  Clinical symptoms of TTP include diarrhea due to ischemic enteritis, abdominal pain, bloody stool, neurological symptoms such as convulsions, visual impairment, and renal impairment. Laboratory findings include changes associated with hemolysis, such as clotted erythrocytes, anemia, thrombocytopenia, serum LDH (lactate dehydrogenase), indirect bilirubin rise, and haptoglobin fall due to thrombus formation. Serum creatinine is also elevated due to kidney damage.

  Known methods for measuring ADAMTS13 activity include SDS-agarose gel electrophoresis (Non-Patent Document 4), FRETS-vWF73 method (Non-Patent Document 5), and the like. In the case of using an immunological technique as a method for measuring ADAMTS13 antigen, anti-ADAMTS13 antibody or a fragment thereof is prepared according to a known method, for example, the method described in International Publication No. 2004/029242. can do.

  In recent years, various measurement methods have been reported as described above, ADAMTS13 activity measurement and ADAMTS13 antigen measurement in various diseases have been performed, and the association between ADAMTS13 and diseases has been reported.

  Conventionally, ADAMTS13 was markedly decreased in most acquired TTP patients, and the cause of this was suspected to be the presence of autoantibodies against ADAMTS13. Matsumoto et al. Analyzed ADAMTS13 and its inhibitor activity in 493 patients with TTP / HUS [hemolytic uremic syndrome] accumulated over the past 7 years. ADAMTS13 activity was observed in 1/3 of all patients. In the remaining 2/3, the activity is not significantly decreased, and the cause is unknown (Non-patent Document 6). Furthermore, it has been reported that even if ADAMTS13 is markedly reduced, there are patients who do not recognize autoantibodies to ADAMTS13. Thus, the mechanism of ADAMTS13 reduction in a patient whose ADAMTS13 is reduced due to causes other than autoantibodies has not yet been clarified. In the previous reports, the presence or absence of autoantibodies is mainly determined by an indirect method of inhibiting ADAMTS13 in normal human plasma. The measurement method for directly detecting autoantibodies against ADAMTS13 is Not yet reported.

  Further, the investigation of the cause of the decrease in ADAMTS13 is considered to be very important because it is closely related to the treatment policy of patients who need early diagnosis and early treatment. As a method for treating congenital TTP, at present, fresh frozen plasma (FFP) is transfused every 2 to 3 weeks, ADAMTS13 supplementation is performed, platelet count is maintained, and onset prevention treatment is performed. Platelet transfusion is contraindicated. For the treatment of acquired TTP, for the purpose of removing the above-mentioned autoantibodies against ADAMTS13 and ultra large vWF multimer (UL-vWFM) from the blood, and supplementing deficient ADAMTS13 Plasma exchange (PE) therapy is the first choice, and FFP infusion is also performed depending on the condition. Prior to the introduction of PE or FFP infusion therapy, the mortality rate was slightly over 80%, and the prognosis was very poor. However, the combined use of these and antiplatelet therapy significantly improved the prognosis. It is almost 90% or more. However, it is known that there are many treatment resistant cases or recurrence cases, and it is predicted that the cause of the decrease in ADAMTS13 is not necessarily limited to that reported so far. In recent years, it has already been reported that hemoglobin in a hemolyzed sample inhibits the activity of ADAMTS13, and a cause of a decrease in activity unrelated to autoantibodies has been shown (Non-patent Document 7). The fact that disrupted erythrocytes and hemolytic anemia are observed as TTP findings, and the fact that the hemoglobin concentration in the sample inhibits ADAMTS13 activity, results in erroneous measurement values being reported in such samples. It is likely to lead to wrong treatment.

G. G. Levy et al., “Nature” (UK), 2001, Vol. 413, p. 488-494. M. Furlan et al., “New England Journal of Medicine” (UK), 1998, 339, p. 1578-1584. Tsai H-M et al., “New England Journal of Medicine” (UK), 1998, 339, p. 1585-1594. M. Furlan et al., “Blood”, (USA), 1997, 89, p. 3097-3103. Kokame K et al., “British Journal of Haematology (UK), 2005, Vol. 129, pp. 930-100. Masanori Matsumoto, “Vascular Medicine” (Japan), 2005, Vol. 6, p. 65-72. J-D Studt et al., “Blood”, (USA), 2005, vol. 105, p. 542-544.

The purpose of the present invention is to analyze the mechanism of the decrease in ADAMTS13 other than autoantibodies or to affect ADAMTS13 measurement, since it is known that there are many treatment resistant cases or recurrence cases even if PE is performed. It is to provide a means for identifying such factors.
Another object is to grasp the dynamics of the true ADAMTS13 in the patient specimen by the above analysis and to provide appropriate treatment options.

  As a result of extensive research, the inventor has measured the amount of ADAMTS13 and the enzyme activity, and in a sample with a low activity and a high antigen, the amount of bilirubin, or the amount of bilirubin and hemoglobin are combined. By analyzing, it was found that the cause of the low ADAMTS13 activity was due to bilirubin, or bilirubin and hemoglobin, and the present invention was completed here.

  That is, the cause of the low value of ADAMTS13 activity in a patient who has been low in ADAMTS13 activity is an autoantibody against ADAMTS13, and plasma exchange (PE) therapy is the best treatment method. It was done. However, in patients with low ADAMTS13 specific activity, the cause of the low value of ADAMTS13 activity is inhibition by hemoglobin or bilirubin, and the treatment option is not conventional PE therapy, but is a method of removing hemoglobin or bilirubin. It is not necessary to use PE, which is expensive and painful for patients, as the first treatment.

According to the present invention, there is provided a cause of a low specific activity of von Willebrand factor cleaving enzyme, characterized by measuring the amount of bilirubin in a subject having a low specific activity of von Willebrand factor cleaving enzyme according to the present invention. It can be solved by the method of determination.
According to a preferred embodiment of the determination method of the present invention, the amount of hemoglobin is further analyzed.

Further, the present invention determines a treatment method for a low specific activity of von Willebrand factor cleaving enzyme, characterized by measuring the amount of bilirubin in a subject having a low specific activity of von Willebrand factor cleaving enzyme. On how to do.
According to a preferred embodiment of the determination method of the present invention, the amount of hemoglobin is further analyzed.

  According to the present invention, by measuring the amount of bilirubin, it can be determined whether the cause of the low specific activity of ADAMTS13 is due to inhibition by bilirubin or bilirubin and hemoglobin. In patients where the specific activity of ADAMTS13 is determined not to be due to autoantibodies to ADAMTS13 but to inhibition by bilirubin or bilirubin and hemoglobin, expensive and painful PE therapy is the first treatment Instead of selecting as a method, another more optimal therapy, such as the bilirubin adsorption method, can be selected.

  As used herein, “von Willebrand factor cleaving enzyme (vWF cleaving enzyme)” specifically cleaves tyrosine (842) and methionine (843) present in the A2 domain of von Willebrand factor (vWF), and ADAMTS13 It is also called a metalloprotease.

In the method of the present invention, the amount of bilirubin and, if desired, further hemoglobin is measured. Bilirubin is a biodegradation product of heme contained in hemoglobin and the like, and is a reddish brown bile pigment. It is a cause of the color of feces and urine of many animals such as humans. It is a kind of tetrapyrrole compound having a structure in which four pyrroles are connected. Although the amount of bilirubin in the blood varies greatly, it increases further greatly in various diseases, so it is an important item in blood tests.
Bilirubin is basically a waste product that is formed after red blood cells die and hemoglobin is degraded. Hemoglobin is decomposed by macrophages, of which heme is further decomposed into Fe ²⁺ , carbon dioxide and biliverdin, and biliverdin is further reduced to bilirubin. A condition with a high total bilirubin (T-Bil) concentration in the blood is called hyperbilirubinemia, a condition with a high direct bilirubin (D-Bil) concentration is called a hyperdirective bilirubinemia, and indirect bilirubin in the blood A condition with high (I-Bil) concentration is called hyperindirect bilirubinemia.

  Adult hemoglobin is a tetramer composed of a total of four subunits, each of which has two polypeptide chains of α and β chains. Each subunit is a combination of a polypeptide portion called globin and one heme portion, and the molecular weight is about 16,000 per piece. The total molecular weight of the hemoglobin molecule is about 64,000 and contains four hemes. Heme is a porphyrin derivative in which a divalent iron atom is coordinated in the center. Here, it combines with oxygen and is transported through the blood to each tissue.

  Test samples that can be used in the method of the present invention include various body fluids that can be measured as biological samples, such as blood, plasma, serum, cell tissue fluid, lymph fluid, thymic fluid, ascites, amniotic fluid, gastric fluid, urine, pancreatic fluid, Bone marrow fluid or saliva can be used, and blood in plasma or serum form is preferred. The plasma is preferably citrate plasma or heparin plasma.

  As long as the subject (subject) to which the method of the present invention can be applied is a subject in which ADAMTS13 is decreased (including a decrease in the amount or enzyme activity of ADAMTS13 or specific activity), or a subject in which a decrease is suspected, The subject is not particularly limited, and is preferably a subject in which the specific activity of ADAMTS13 is reduced or a subject suspected of being lowered, and more preferably a subject in which the specific activity of ADAMTS13 is reduced.

In this specification, “a subject having a low specific activity of ADAMTS13” means that when a sample is collected from the subject and the amount of ADAMTS13 (antigen amount) and the enzyme activity in the sample are measured, the enzyme activity is low. It means a target in which the antigen amount is higher than the value expected from the enzyme activity and the antigen amount and enzyme activity are different. In the subject patient in the present invention, both the ADAMTS13 antigen amount and the enzyme activity are lower than the respective normal values, but the enzyme activity is significantly reduced as compared with the decrease in the antigen amount, and the antigen amount and the activity value are different. ing.
In this specification, “specific activity” means a value obtained by dividing enzyme activity (relative activity value) by the amount of antigen (relative value). The enzyme activity and the antigen amount are relative values with respect to normal values.
The normal value of the specific activity of ADAMTS13 is expected to vary depending on various conditions such as the underlying disease, sex, age, etc., but those skilled in the art can appropriately select an appropriate population corresponding to the subject. The normal value range can be determined by performing statistical processing on the data obtained from the population. For example, the specific activity is usually in the range of about 0.9 to 1.1 in a healthy person or a patient whose antigen amount and activity value are not different. In this specification, “the specific activity of ADAMTS13 is low” means that the specific activity is less than 0.8.

  In the method of the present invention, when a sample is collected from a patient suspected of lowering ADAMTS13, and the amount and enzyme activity of von Willebrand factor cleaving enzyme are measured, the sample has a low activity and the antigen deviates to a higher value than the activity. Then, by further measuring bilirubin, or bilirubin and hemoglobin, the cause of the low value of von Willebrand factor cleaving enzyme activity can be grasped, and further, an appropriate treatment method can be determined.

  In the method of the present invention, the method for analyzing the ADAMTS13 concentration is particularly limited as long as the ADAMTS13 amount can be quantitatively or semi-quantitatively determined, or the presence or absence of ADAMTS13 can be determined. For example, an immunological method using an anti-ADAMTS13 antibody or a fragment thereof (for example, enzyme immunoassay, latex agglutination immunoassay, chemiluminescence immunoassay, fluorescent antibody method, radioimmunoassay, immunoprecipitation method , Immunohistochemical staining, Western blot, etc.), biochemical techniques (for example, enzymatic measurement methods), or molecular biological techniques for measuring the amount of mRNA.

  When an immunological technique is used as an analysis method for ADAMTS13, the anti-ADAMTS13 antibody can be prepared according to a known method, for example, the method described in International Publication No. 2004/029242. The physical measurement can also be performed, for example, according to the method described in International Publication No. 2004/029242.

As a method for measuring the ADAMTS13 concentration, an immunological method is preferable from the viewpoint of sensitivity and simplicity. Here, the immunological method is a method of analyzing ADAMTS13 using, for example, an ELISA method, a latex method, or an immunochromatographic method using an antibody against ADAMTS13. Examples of immunological methods include competitive methods for labeling ADAMTS13, sandwich methods for labeling antibodies, latex bead methods for observing aggregation of antibody-coated beads, or antibodies bound to colored particles such as gold colloids. Although there are various methods such as a method to be used, a method using an antibody against ADAMTS13 is included in a preferred embodiment of the present invention. The antibody may be a monoclonal antibody or a polyclonal antibody. Antibody fragments such as Fab, Fab ′, F (ab ′) ₂ , or Fv can also be used.

  The enzyme activity of ADAMTS13 can be measured by, for example, a method using SDS-agarose electrophoresis [M. Furlan et al., “Blood”, (USA), 1997, Vol. 89, p. 3097-3103], ELISA method using a recombinant antigen of the AWF domain of vWF, which is a substrate of ADAMTS13 [Whitelock JL et al., "Journal of Thrombosis and Haemostasis", (UK), 2004, Vol.2, 485-491], or a synthetic peptide corresponding to residue 73 of Asp1596-Arg1668 in the A2 domain of vWF which is a substrate of ADAMTS13 [2- (N-methylamino) benzoyl, Nma] And a method using a quenching fluorescent substrate FRETS-VWF73 introduced with a quenching group (2,4-dinitrophenyl, Dnp) [Kokame K et al., “British Journal of Hematology” (UK), 2005, No. 129, 93-100]. Further, the method described in WO 2006/123789, specifically, (1) a test sample that may contain ADAMTS13, and an immobilized substrate in which vWF or a fragment thereof is bound to an insoluble carrier (2) a step of separating the liquid from the insoluble carrier, and (3) vWF remaining in the insoluble carrier or a fragment thereof, and / or vWF in the liquid released from the insoluble carrier. It can be measured by an analysis method including a step of analyzing fragments.

The bilirubin to be measured in the method of the present invention can be any of total bilirubin, direct bilirubin, or indirect bilirubin, preferably total bilirubin. Bilirubin that has undergone glucuronidation in the liver is called direct bilirubin, bilirubin before receiving glucuronidation is called indirect bilirubin, and the sum of both is called total bilirubin.
Regarding the method for measuring the bilirubin concentration, for example, a method using an enzymatic method using bilirubin oxidase [kurosaka.k et al., “Clinica Chimica Acta”, (USA), 1998, 269 Volume, p. 125-136] or a method using the vanadic acid method [Tokuda.K, et al., “Nippon Clinical Chemistry”, (Japan), 1993, Vol. 22, p. 116-122].

  Regarding the method for measuring the hemoglobin concentration, a widely used automatic blood count measuring instrument is preferable in terms of simplicity and reproducibility. As the measurement principle, for example, cyanmethemoglobin method [edited by Shiro Miwa, edited by Clinical Laboratory Technology, 3, 1972, p. 241-275], SLS-hemoglobin method [Oshiro, et al., Clinical Pathology, 1981, Vol. 29, p. 203-209] and the like, but the SLS-hemoglobin method is preferable in view of the problem of waste liquid.

  In the method of the present invention, the cause of the low specific activity of ADAMTS13 can be determined by measuring the amount of bilirubin (and, if desired, the amount of hemoglobin), and an optimal treatment method based on the recognized cause (that is, The method of returning the reduced specific activity of ADAMTS13 to a normal value) can be determined.

  In the method of the present invention, when the amount of bilirubin, or the amount of bilirubin and the amount of hemoglobin is higher than the normal value range of a healthy person (for example, exceeding a threshold value), the cause of the low specific activity of ADAMTS13 is bilirubin, Alternatively, it can be determined to be due to inhibition by bilirubin and hemoglobin. In this case, for example, bilirubin removal by a bilirubin adsorption method can be selected as an optimal treatment method.

In the method of the present invention, the determination can be performed by collecting specimens from healthy subjects and subjects, measuring the amount of each bilirubin (and, if desired, further, the amount of hemoglobin), and then comparing the measured values. Usually, it is preferable to previously determine a normal value range or a determination threshold value for the amount of bilirubin (and further, if necessary, the amount of hemoglobin) using a sample collected from a healthy person. When the normal value range or the threshold for determination is determined in advance, the determination in the subject can be performed only by analyzing the amount of bilirubin (and if necessary, the amount of hemoglobin) with respect to the subject to be predicted. . The normal value range or the threshold for determination is expected to change depending on various conditions such as the underlying disease, sex, age, etc., but those skilled in the art will appropriately select an appropriate population corresponding to the subject. Thus, by performing statistical processing on the data obtained from the population, the normal value range or the determination threshold value can be determined.
For example, regarding the total bilirubin concentration, the value that can be regarded as abnormal is usually 1 mg / dL or more, and in the present invention, it is 3 mg / dL or more, and further 10 mg / dL or more.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<< Example 1: In vitro inhibition by bilirubin, or bilirubin and hemoglobin >>
Bilirubin (Sysmex) is added to normal human plasma to 0,2,10,20,40 mg / dL and hemoglobin (Sysmex) to 0,10,50,250,500 mg / dL, and the FRETS-vWF73 method ADAMTS13 activity was measured by [Peptide Institute, Inc.]. In addition, bilirubin F (indirect bilirubin) and bilirubin C (direct bilirubin) were used as bilirubin.

  The results are shown in FIGS. ADAMTS13 activity decreased with increasing concentrations of both hemoglobin and bilirubin. Bilirubin 40 mg / dL decreased to 16% compared to plasma without bilirubin (0 mg / dL), and decreased to 50% compared with plasma without hemoglobin (0 mg / dL) in the presence of 500 mg / dL hemoglobin. .

<< Example 2: Relationship between presence / absence of autoantibodies in patients and measured values of various markers >>
3 specimens (samples 1 to 3) with suspected TTP in cases of periodic neutropenia-associated sepsis, 1 patient with suspected TTP who developed liver tumor rupture and disseminated intravascular coagulation syndrome (DIC) ( Sample 4) Using 3.8% citrate plasma collected from one ticlopidine-administered patient (sample 5), ADAMTS13 activity (Act) was determined by the FRETS-vWF73 method [Peptide Institute, Inc.] ADAMTS13 antigen level. (Ag) was measured by ADAMTS13 ELISA kit [Mitsubishi Chemical Yatron Co., Ltd.]. Hemoglobin (Hb) in blood was measured with Sysmex CA1500, and total bilirubin (T-Bil) in patient serum was measured with HITACHI-71010S.

  30 μL of each specimen and 40 μL of protein G sepharose were mixed and reacted overnight at 4 ° C. with stirring. After centrifugation, the supernatant was collected and used as an IgG-deficient fraction. Next, 50 μL of 0.1 mol / L glycine (pH 2.7) was added to the precipitated protein G sepharose and allowed to react at room temperature for 3 minutes. After centrifugation, the supernatant was collected and 2 mol / L Tris buffer ( pH 9.5) IgG fraction was obtained by adding 1 μL and returning the pH to neutral. The IgG-deficient fraction or the IgG fraction 6 μL was mixed with 6 μL of healthy human plasma, respectively, and reacted at 37 ° C. for 2 hours. After the reaction, 4 μL of the mixed solution was used to examine the inhibition rate of healthy human plasma by an activity measurement method using FRETS-vWF73.

  The results are shown in Table 1. In specimen 4, a significant decrease in ADAMTS13 specific activity (0.19) and a high total bilirubin value (33.96 mg / dL) were observed. The ADAMTS13 inhibition rate of healthy human plasma by IgG purified from the sample was 50% or less, and did not strongly support the presence of autoantibodies.

<Table 1>
ADAMTS13
Specimen Antigen level (Ag) Activity (Act) Specific activity Hb T-Bil Platelet inhibition rate
Number (%) (%) (Act / Ag) (g / dL) (mg / dL) 10 ³ / μL (%)
1 53.7 25.4 0.47 8.2 2.58 529 44
2 99.0 32.6 0.33 7.7 7.28 148 14
3 85.2 61.8 0.73 8.2 2.46 148 24
4 59.2 11.0 0.19 9.7 33.96 21 32
5 4.1 6.5 1.59 5.6 2.22 7 13

<< Example 3: Effect of bilirubin dye decolorization with bilirubin oxidase on ADAMTS13 activity measurement >>
In order to examine whether the inhibition of ADAMTS13 activity by a bilirubin specimen has an influence on the fluorescence measurement by the dye of bilirubin, normal human plasma is adjusted to 0, 2.5, 10, 20, 40 mg / dL of bilirubin (Sysmex). After adding 3 U bilirubin oxidase and 1 mg / mL sodium cholate to the sample added to the sample and incubating at 37 ° C. for 1 hour to decompose the bilirubin dye, the activity was measured by FRETS-vWF73. In addition, as bilirubin, what mixed equal amounts of bilirubin F and bilirubin C was used.

  The obtained results are shown in FIG. 3 together with the results shown in FIG. ADAMTS13 activity is reduced to 40% due to the effects of bilirubin oxidase and cholic acid even in a sample to which no bilirubin is added. However, ADAMTS13 activity is reduced due to the increase in bilirubin concentration. Was not due to bilirubin pigment.

"Example 4: Non Study of ADAMST13 active retraction of bilirubin analytes by CaCl ₂ extenders"
In order to investigate whether the inhibition of ADAMTS13 activity by a bilirubin sample is due to the inhibition of the cleavage activity of ADAMTS13, which is a metalloprotease, by chelating Ca ions by bilirubin, in the buffer used at the time of measuring FRETS-vWF The ADAMTS13 activity was measured when the concentration of CaCl ₂ was increased from the current 25 mmol / L to 125 mmol / L. Recently, ADAMTS13 activity has been reported to be enhanced by Ca ions and Zn ions [PJ Anderson et al., “JBC (JBC)”, (USA), 2006, Vol. 281, p. 850-857], a system to which 0.1 mmol / L ZnCl ₂ was added was also examined. The results are shown in FIG. When the CaCl ₂ concentration was increased, the activity of the bilirubin 0 mg / dL sample was also reduced, but the ADAMTS13 activity inhibition by the high bilirubin concentration sample could not be recovered.

<< Example 5: Inhibition by bilirubin of activity by SDS-agarose electrophoresis >>
Bilirubin (Sysmex) was added to normal human plasma at 0, 10, 20, 40 mg / dL, and SDS-agarose gel electrophoresis [M. Furlan et al., “Blood” (USA), 1997, 89, p. 3097-3103. ] To measure ADAMTS13 activity. The results are shown in FIG. Similarly to the FRETS-vWF73 method using 73 peptides in the A2 domain of vWF, activity inhibition by bilirubin was also observed in the activity measurement method by SDS-agarose electrophoresis using vWF purified from plasma as a substrate. These facts revealed that bilirubin acts as an inhibitor of ADAMTS13 activity regardless of the type of ADAMTS13 substrate and the measurement method.

  The present invention can be applied to grasp the cause of the low ADAMTS13 specific activity.

It is a graph which shows the result of inhibition of ADAMTS13 activity dependent on the bilirubin concentration added to the healthy subject sample. It is a graph which shows the result of inhibition of ADAMTS13 activity dependent on the hemoglobin concentration added to the healthy subject sample. It is a graph which shows the influence which it has on ADAMTS13 activity when the healthy subject sample which added bilirubin is oxidized by bilirubin oxidase treatment. In inhibiting the bilirubin concentration-dependent ADAMTS13 activity is a graph showing the effect of CaCl ₂ added. It is a graph which shows the result of inhibition of ADAMTS13 activity by SDS-agarose electrophoresis method dependent on the bilirubin concentration added to the healthy subject sample.

Claims (4)

  A method for determining the cause of a low specific activity of von Willebrand factor cleaving enzyme, comprising measuring the amount of bilirubin in a subject having a low specific activity of von Willebrand factor cleaving enzyme.   Furthermore, the method of Claim 1 which measures the amount of hemoglobin.   A method for determining a treatment method for a low specific activity of von Willebrand factor cleaving enzyme, comprising measuring the amount of bilirubin in a subject having a low specific activity of von Willebrand factor cleaving enzyme.   Furthermore, the method of Claim 3 which measures the amount of hemoglobin.

Images